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Many neurodegenerative conditions are characterized by the deposition of protein aggregates (mainly amyloid-like) in the central nervous system (CNS). In post-mitotic CNS cells protein aggregation causes cytotoxicity by interfering with various cellular functions. Mutations in different genes may directly cause protein aggregation. However, genetic factors together with aging may contribute to the onset of protein aggregation also by affecting cellular degradative functions, in particular the autophagy-lysosomal pathway (ALP). Increasing body of evidence show that ALP dysfunction and protein aggregation are functionally interconnected and induce each other during neurodegenerative processes. We will summarize the findings supporting these concepts by focusing on lysosomal storage diseases (LSDs), a class of metabolic inherited conditions characterized by global lysosomal dysfunction and often associated to a severe neurodegenerative course. We propose a model by which the inherited lysosomal defects initiate aggregate-prone protein deposition, which, in turns, worsen ALP degradation function, thus generating a vicious cycle, which boost neurodegenerative cascades.

Lysosomal storage disorders characterized by altered metabolism of heparan sulfate, including Mucopolysaccharidosis (MPS) III and MPS-II, exhibit lysosomal dysfunctions leading to neurodegeneration and dementia in children. In lysosomal storage disorders, dementia is preceded by severe and therapy-resistant autistic-like symptoms of unknown cause. Using mouse and cellular models of MPS-IIIA, we discovered that autistic-like behaviours are due to increased proliferation of mesencephalic dopamine neurons originating during embryogenesis, which is not due to lysosomal dysfunction, but to altered HS function. Hyperdopaminergia and autistic-like behaviours are corrected by the dopamine D1-like receptor antagonist SCH-23390, providing a potential alternative strategy to the D2-like antagonist haloperidol that has only minimal therapeutic effects in MPS-IIIA. These findings identify embryonic dopaminergic neurodevelopmental defects due to altered function of HS leading to autistic-like behaviours in MPS-II and MPS-IIIA and support evidence showing that altered HS-related gene function is causative of autism. Lysosomal storage disorders, characterized by altered metabolism of heparan sulfate, cause autistic symptoms followed by dementia in children. Here, the authors show that embryonic dopaminergic neurodevelopmental defects due to altered function of heparan sulfate cause autistic behaviours in mice.

The function of lysosomes relies on the ability of the lysosomal membrane to fuse with several target membranes in the cell. It is known that in lysosomal storage disorders (LSDs), lysosomal accumulation of several types of substrates is associated with lysosomal dysfunction and impairment of endocytic membrane traffic. By analysing cells from two severe neurodegenerative LSDs, we observed that cholesterol abnormally accumulates in the endolysosomal membrane of LSD cells, thereby reducing the ability of lysosomes to efficiently fuse with endocytic and autophagic vesicles. Furthermore, we discovered that soluble N‐ethylmaleimide‐sensitive factor attachment protein (SNAP) receptors (SNAREs), which are key components of the cellular membrane fusion machinery are aberrantly sequestered in cholesterol‐enriched regions of LSD endolysosomal membranes. This abnormal spatial organization locks SNAREs in complexes and impairs their sorting and recycling. Importantly, reducing membrane cholesterol levels in LSD cells restores normal SNARE function and efficient lysosomal fusion. Our results support a model by which cholesterol abnormalities determine lysosomal dysfunction and endocytic traffic jam in LSDs by impairing the membrane fusion machinery, thus suggesting new therapeutic targets for the treatment of these disorders. Lysosomal storage disorders involve lysosomal dysfunction and defective endocytic membrane trafficking. However, the underlying mechanism(s) remain largely unclear. In this study, Andrea Ballabio et al. reveal that cholesterol accumulates in the endolysosomal system of LSD cells and interferes with the activity of the lysosomal SNARE membrane fusion machinery

Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders caused by a deficiency in lysosomal enzymes catalyzing the stepwise degradation of glycosaminoglycans (GAGs). The current therapeutic strategies of enzyme replacement therapy and allogeneic hematopoietic stem cell transplantation have been reported to reduce patient morbidity and to improve their quality of life, but they are associated with persistence of residual disease burden, in particular at the neurocognitive and musculoskeletal levels. This indicates the need for more efficacious treatments capable of effective and rapid enzyme delivery to the affected organs, especially the brain and the skeleton. Gene therapy (GT) strategies aimed at correcting the genetic defect in patient cells could represent a significant improvement for the treatment of MPS when compared with conventional approaches. While in-vivo GT strategies foresee the administration of viral vector particles directly to patients with the aim of providing normal complementary DNA to the affected cells, ex-vivo GT approaches are based on the ex-vivo transduction of patient cells that are subsequently infused back. This review provides insights into the state-of-art accomplishments made with in vivo and ex vivo GT-based approaches in MPS and provide a vision for the future in the medical community.

Lysosomal storage disorders (LSDs) are inherited diseases characterized by lysosomal dysfunction and often showing a neurodegenerative course. There is no cure to treat the central nervous system in LSDs. Moreover, the mechanisms driving neuronal degeneration in these pathological conditions remain largely unknown. By studying mouse models of LSDs, we found that neurodegeneration develops progressively with profound alterations in presynaptic structure and function. In these models, impaired lysosomal activity causes massive perikaryal accumulation of insoluble α‐synuclein and increased proteasomal degradation of cysteine string protein α (CSPα). As a result, the availability of both α‐synuclein and CSPα at nerve terminals strongly decreases, thus inhibiting soluble NSF attachment receptor (SNARE) complex assembly and synaptic vesicle recycling. Aberrant presynaptic SNARE phenotype is recapitulated in mice with genetic ablation of one allele of both CSPα and α‐synuclein. The overexpression of CSPα in the brain of a mouse model of mucopolysaccharidosis type IIIA, a severe form of LSD, efficiently re‐established SNARE complex assembly, thereby ameliorating presynaptic function, attenuating neurodegenerative signs, and prolonging survival. Our data show that neurodegenerative processes associated with lysosomal dysfunction may be presynaptically initiated by a concomitant reduction in α‐synuclein and CSPα levels at nerve terminals. They also demonstrate that neurodegeneration in LSDs can be slowed down by re‐establishing presynaptic functions, thus identifying synapse maintenance as a novel potentially druggable target for brain treatment in LSDs. Synopsis Neurodegeneration associated with lysosomal dysfunction in lysosomal storage disorders (LSDs) may be linked to impaired presynaptic maintenance initiated by a reduction in α‐synuclein and CSPα levels at nerve terminals. α‐Synuclein and cysteine string protein (CSP)α are two key chaperones, which ensure efficient SNARE complex formation and synaptic vesicle recycling by maintaining physiological SNARE levels at nerve terminals. Lysosomal dysfunction causes both the accumulation of undegraded α‐synuclein in insoluble aggregates perikarya and the enhanced proteasomal degradation of CSPα. The unbalanced proteostasis results in the simultaneous depletion of α‐synuclein and CSPα at nerve terminals. This in turn caused a reduction in presynaptic SNARE levels, thus leading to synaptic dysfunction. Viral‐mediated CSPα overexpression in a mouse model of mucopolysaccharidosis type IIIA mice (a severe neurodegenerative LSD) exerted a protective action against neurodegeneration by re‐establishing efficient SNARE complex formation and improving presynaptic function. Graphical Abstract Neurodegeneration associated with lysosomal dysfunction in lysosomal storage disorders (LSDs) may be linked to impaired presynaptic maintenance initiated by a reduction in α‐synuclein and CSPα levels at nerve terminals.

In vivo genetic engineering has recently shown remarkable potential as a novel effective treatment for an ever-growing number of diseases, as also witnessed by the recent marketing authorization of several in vivo gene therapy products. In vivo genetic engineering comprises both viral vector-mediated gene transfer and the more recently developed genome/epigenome editing strategies, as long as they are directly administered to patients. Here we first review the most advanced in vivo gene therapies that are commercially available or in clinical development. We then highlight the major challenges to be overcome to fully and broadly exploit in vivo gene therapies as novel medicines, discussing some of the approaches that are being taken to address them, with a focus on the nervous system and liver taken as paradigmatic examples.

Mucopolysaccharidoses type IIIA (MPS‐IIIA) is a neurodegenerative lysosomal storage disorder (LSD) caused by inherited defects of the sulphamidase gene. Here, we used a systemic gene transfer approach to demonstrate the therapeutic efficacy of a chimeric sulphamidase, which was engineered by adding the signal peptide (sp) from the highly secreted iduronate‐2‐sulphatase (IDS) and the blood‐brain barrier (BBB)‐binding domain (BD) from the Apolipoprotein B (ApoB‐BD). A single intravascular administration of AAV2/8 carrying the modified sulphamidase was performed in adult MPS‐IIIA mice in order to target the liver and convert it to a factory organ for sustained systemic release of the modified sulphamidase. We showed that while the IDS sp replacement results in increased enzyme secretion, the addition of the ApoB‐BD allows efficient BBB transcytosis and restoration of sulphamidase activity in the brain of treated mice. This, in turn, resulted in an overall improvement of brain pathology and recovery of a normal behavioural phenotype. Our results provide a novel feasible strategy to develop minimally invasive therapies for the treatment of brain pathology in MPS‐IIIA and other neurodegenerative LSDs. →See accompanying article emmm.201302668 Graphical Abstract Gene transfer of a liver‐targeted sulfamidase engineered for increased secretion and blood brain barrier permeability, effectively ameliorates overall brain pathology and behavioural phenotype in treated Mucopolysaccharidosis (MPS) type IIIA mice.

Mucopolysaccharidosis type IIIA (MPS-IIIA, Sanfilippo A) is one of the most severe lysosomal storage disorder (LSD) caused by the inherited deficiency of sulfamidase, a lysosomal sulfatase enzyme involved in the stepwise degradation of heparan sulfates (HS). MPS-IIIA patients show multisystemic problems, including a strong impairment of central nervous system (CNS), mild somatic involvement, and ocular manifestations that result in significant visual impairment. Despite the CNS and somatic pathology have been well characterized, studies on visual system and function remain partially explored. Here, we characterized the retina morphology and functionality in MPS-IIIA mouse model and analyzed how the SGSH deficiency affects the autophagic flux. MPS-IIIA mice exhibited a progressive retinal dystrophy characterized by significant alterations in visual function. The photoreceptor degeneration was associated with HS accumulation and a block of autophagy pathway. These events caused a reactive microgliosis, and a development of apoptotic processes in MPS-IIIA mouse retina. Overall, this study provides the first phenotypic spectrum of retinal disorders in MPS-IIIA and significantly contributes for diagnosis, counseling, and potential therapies development.

Key Points Lysosomes are cellular organelles involved in the degradation and recycling of cellular waste. Extracellular and intracellular materials to be degraded reach the lysosome via endocytosis and autophagy, respectively. Lysosomes are also involved in secretion and plasma membrane repair, by fusing to the plasma membrane in a process termed lysosomal exocytosis. Lysosomal function is performed by lumenal hydrolases that are responsible for substrate digestion and by membrane-associated proteins that handle trafficking of materials into and out of the lysosome. A complex machinery, which includes the kinase complex mammalian target of rapamycin complex 1 (mTORC1, a major regulator of cell growth), the vesicular ATPase complex and additional complexes, is located on the lysosomal surface and is devoted to sensing the nutrient content of the lysosome. This complex is called the lysosomal nutrient sensing (LYNUS) machinery. Most genes encoding lysosomal proteins belong to a gene network termed CLEAR (coordinated lysosomal expression and regulation), and they are transcriptionally regulated by transcription factor EB (TFEB), the master regulator for lysosomal biogenesis. Using this regulatory mechanism, cells can adapt lysosomal function to respond to environmental cues. The activity of TFEB is induced following starvation, by both transcriptional autoregulation and a phosphorylation-dependent mechanism. Once activated, TFEB mediates the starvation response by activating lipid catabolism via the regulation of the master lipid metabolism genes PPAR α (peroxisome proliferator-activated receptor-α) and PGC1α (PPARγ co-activator 1α). TFEB regulation and function are conserved in worms. Lysosomal and autophagy dysfunction occurs both in lysosomal storage diseases (LSDs) and in common neurodegenerative diseases, resulting in defective cellular clearance and the accumulation of toxic material. Thus, TFEB-mediated induction of cellular clearance may represent an attractive therapeutic strategy for these disorders. As well as degrading and recycling cellular waste, lysosomes are involved in secretion, plasma membrane repair, signalling and energy metabolism. The identification of transcription factor EB (TFEB) as a central regulator of lysosomal biogenesis and autophagy provides insight into how lysosomes adapt to environmental cues, and targeting TFEB may be a promising therapeutic strategy for modulating lysosomal function in disease. For a long time, lysosomes were considered merely to be cellular 'incinerators' involved in the degradation and recycling of cellular waste. However, now there is compelling evidence indicating that lysosomes have a much broader function and that they are involved in fundamental processes such as secretion, plasma membrane repair, signalling and energy metabolism. Furthermore, the essential role of lysosomes in autophagic pathways puts these organelles at the crossroads of several cellular processes, with significant implications for health and disease. The identification of a master regulator, transcription factor EB (TFEB), that regulates lysosomal biogenesis and autophagy has revealed how the lysosome adapts to environmental cues, such as starvation, and targeting TFEB may provide a novel therapeutic strategy for modulating lysosomal function in human disease.

Cerebrospinal fluid administration of recombinant adeno-associated viral (rAAV) vectors has been demonstrated to be effective in delivering therapeutic genes to the central nervous system (CNS) in different disease animal models. However, a quantitative and qualitative analysis of transduction patterns of the most promising rAAV serotypes for brain targeting in large animal models is missing. Here, we characterize distribution, transduction efficiency, and cellular targeting of rAAV serotypes 1, 2, 5, 7, 9, rh.10, rh.39, and rh.43 delivered into the cisterna magna of wild-type pigs. rAAV9 showed the highest transduction efficiency and the widest distribution capability among the vectors tested. Moreover, rAAV9 robustly transduced both glia and neurons, including the motor neurons of the spinal cord. Relevant cell transduction specificity of the glia was observed after rAAV1 and rAAV7 delivery. rAAV7 also displayed a specific tropism to Purkinje cells. Evaluation of biochemical and hematological markers suggested that all rAAV serotypes tested were well tolerated. This study provides a comprehensive CNS transduction map in a useful preclinical large animal model enabling the selection of potentially clinically transferable rAAV serotypes based on disease specificity. Therefore, our data are instrumental for the clinical evaluation of these rAAV vectors in human neurodegenerative diseases.